Hematological malignancies are a major public health problem. It has been estimated that in the year 2000, more than 50,000 new cases of non-Hodgkin's lymphoma and more than 30,000 new cases of leukemia occurred in the United States (Greenlee, R. T. et al., CA Cancer J. Clin., 50:7-33 (2000)) and more than 45,000 deaths were expected from these diseases. Many more patients live with chronic disease-related morbidity. Unfortunately, in a high percentage of patients, conventional therapies are not able to induce long term complete remissions.
In the past several years immunotoxins have been developed as an alternative therapeutic approach to treat these malignancies. Immunotoxins were originally composed of an antibody chemically conjugated to a plant or a bacterial toxin. The antibody binds to the antigen expressed on the target cell and the toxin is internalized causing cell death by arresting protein synthesis and inducing apoptosis (Brinkmann, U., Mol. Med. Today, 2:439-446 (1996)).
Hematological malignancies are an attractive target for immunotoxin therapies because tumor cells are easily accessible and the target antigens are highly expressed (Kreitman, R. J. and Pastan, I., Semin. Cancer Biol., 6:297-306 (1995)). One of these antigens is CD25. A clinical trial with immunotoxin LMB-2 (anti-Tac(Fv)-PE38) that targets CD25 showed that the agent was well tolerated and that it had substantial anti-tumor activity (Kreitman, R. J. et al., Blood, 94:3340-3348 (1999); Kreitman, R. J. et al., J. Clin. Oncol., 18:16222-1636 (2000)). A complete response was observed in one patient with Hairy Cell Leukemia and partial responses were observed in patients with Hairy Cell Leukemia, chronic lymphocytic leukemia, cutaneous T cell lymphoma, Hodgkins disease and adult T cell leukemia.
Another antigen that has been used as an immunotoxin target is CD22, a lineage-restricted B cell antigen expressed in 60-70% of B cell lymphomas and leukemias. CD22 is not present on the cell surface in the early stages of B cell development and is not expressed on stem cells (Tedder, T. F. et al., Annu. Rev. Immunol., 5:481-504 (1997)). Clinical trials have been conducted with an immunotoxin containing an anti-CD22 antibody, RFB4, or its Fab fragment, coupled to deglycosylated ricin A. In these trials, substantial clinical responses have been observed; however, severe and in certain cases fatal, vascular leak syndrome was dose limiting (Sausville, E. A. et al., Blood, 85:3457-3465 (1995); Amlot, P. L. et al., Blood, 82:2624-2633 (1993); Vitetta, E. S. et al., Cancer Res., 51:4052-4058 (1991)).
As an alternative approach, the RFB4 antibody was used to make a recombinant immunotoxin in which the Fv fragment in a single chain form is fused to a 38 kDa truncated form of Pseudomonas exotoxin A (PE38, SEQ ID NO:22). PE38 contains the translocating and ADP ribosylating domains of PE but not the cell-binding portion (Hwang, J. et al., Cell, 48:129-136 (1987)). RFB4 (Fv)-PE38 is cytotoxic towards CD22-positive cells (Mansfield, E. et al., Biochem. Soc. Trans., 25:709-714 (1997)). To stabilize the single chain Fv immunotoxin and to make it more suitable for clinical development, cysteine residues were engineered into framework regions of the VH and VL (Mansfield, E. et al., Blood, 90:2020-2026 (1997)) generating the molecule RFB4 (dsFv)-PE38.
RFB4 (dsFv)-PE38 is able to kill leukemic cells from patients and induced complete remissions in mice bearing lymphoma xenografts (Kreitman, R. J. et al., Clin. Cancer Res., 6:1476-1487 (2000); Kreitman, R. J. et al., Int. J. Cancer, 81:148-155 (1999)). RFB4 (dsFv)-PE38 (BL22) was evaluated in a phase I clinical trial at the National Cancer Institute in patients with hematological malignancies. Sixteen patients with purine analogue resistant hairy cell leukemia were treated with BL22 and eleven (86%) achieved complete remissions.
These results show that BL22 is the first agent that is able to induce high complete remission rate in patients with purine analogue-resistant HCL and establish the concept that immunotoxins can produce clinical benefit to patients with advanced malignancies (Kreitman, R. J., et al., N Engl J Med, 345(4):241-7 (2001)).
HA22 is a recently developed, improved form of BL22. To produce this immunotoxin, the binding region of antibody RFB4 was mutated and antibody phage display was used to isolate mutant phage that bound better to CD22 because of mutations in CDR3 of the heavy chain. In HA22, residues SSY in the CDR3 of the antibody variable region heavy chain (“VH”) were mutated to THW. Compared to its parental antibody, RFB4, HA22 has a 5-10-fold increase in cytotoxic activity on various CD22-positive cell lines and is up to 50 times more cytotoxic to cells from patients with CLL and HCL (Salvatore, G., et al., Clin Cancer Res, 8(4):995-1002 (2002); see also, co-owned application PCT/US02/30316, International Publication WO 03/027135).
BL22 appears to work well on malignancies, such as HCL, which express significant amounts of CD22. It showed much less activity, however, in chronic lymphocytic leukemia (CLL), in which the cells express only small amounts of CD22. As noted above, HA22-based immunotoxin is much more cytotoxic to cells from persons with CLL than is BL22. Given the low density of CD22 on CLL cells, however, it would be desirable to improve targeting to CLL cells further by developing antibodies with even greater affinity to CD22 than that of HA22.
Unfortunately, the factors that influence binding affinity are multifaceted and obtaining mutant scFvs with improved affinity is not trivial. Although antibody-antigen crystal structure can suggest which residues are involved in binding, atomic resolution structural data are not available for most antibodies. Moreover, even when such data is available it cannot generally be predicted which residues and which mutations will result in an antibody with increased antigen binding activity.
Even if immunotoxins bind tightly to the surface of targeted cells, however, death of the targeted cell is not assured. Commonly used toxins (e.g., diphtheria toxin, gelonin, ricin, and PE) act at the ribosomal level to inactivate protein synthesis. Thus, the toxin must be correctly routed to the ribosome for cell death to occur. For immunotoxins targeted to specific cell-surface receptors, this involves receptor-mediated endocytosis into an appropriate intracellular vesicle, followed by translocation of the toxin across the vesicular membrane to the cytosol. Inefficient intracellular trafficking, e.g, immunotoxins traveling to lysosomes, results in a large reduction in utilization of the targeted toxin (Thrush, G. R., et al., Annu Rev Immunol, 14:49-71 (1996)).
Thus, in addition to increasing the affinity of antibodies such as BL22 or HA22 to CD22, another way to increase the cytotoxicity of immunotoxins to CLL cells would be to increase the cytotoxicity of the toxin moiety. As noted above, a clinical trial of BL22 used as the toxic moiety a form of Pseudomonas exotoxin A (“PE”) truncated to reduce non-specific toxicity, and PE has been used in clinical trials with other targeting agents. Given PE's utility in therapeutic agents, it would be useful to further improve PE's toxicity. But the complicated manner in which PE exerts its toxicity renders improving that toxicity problematic.
Based on the crystallographic structure of PE (Allured, V. S., et al., Proc Natl Acad Sci USA, 83(5):1320-4 (1986)) and many functional studies, BL22 is thought to kill target cells in the circulation by the following steps. First, in the circulation, the carboxy terminal lysine residue is removed (Hessler, J. L., et al., Biochemistry, 36(47):14577-82 (1997)). Next, the Fv portion of the immunotoxin binds to CD22 on the surface of the target cell, and the molecule is internalized into the endocytic compartment, where the protease furin cleaves the toxin between amino acids 279 and 280 of PE (Chiron, M. F., et al., J Biol Chem, 269(27):18167-76 (1994); Ogata, M., et al., J Biol Chem, 265(33):20678-85 (1990)). Subsequently, the disulfide bond linking cysteines at positions 265 and 287 is reduced producing two fragments. Then the REDL (SEQ ID NO:6) sequence on the carboxyl terminal fragment binds to the KDEL (SEQ ID NO:5) recycling receptor and the fragment containing part of domain 2 and all of domain 3 is transported from the trans-reticular Golgi to the endoplasmic reticulum (ER) (Kreitman, R. J., et al., Semin Cancer Biol, 6(5):297-306 (1995)). Once there, amino acids 280-313 somehow facilitate translocation of the toxin into the cytosol, probably taking advantage of preexisting pores in the ER (Theuer, C. P., et al., Proc Natl Acad Sci USA, 90(16):7774-8 (1993); Theuer, C., et al., Biochemistry, 33(19):5894-900 (1994)). In the cytosol, the ADP ribosylation activity located within domain III of PE catalytically inactivates elongation factor 2, inhibiting protein synthesis and leading to cell death.
The present invention provides solutions to some of these difficult problems.